Plasma membrane-embedded receptors serve as biomarkers for many human diseases and represent more than 50% of drug targets (Molek et al., 2011. Peptide phage display as a tool for drug discovery: targeting membrane receptors. Molecules 16, 857-887). There is a demand in research, diagnostics, and therapeutics for affinity reagents that recognize this class of molecules, but antibodies against membrane proteins can be difficult to obtain. This is because these proteins strongly rely on their native environment for structural integrity, and they often contain glycosylation and other post-translational modifications. Affinity reagents, such as antibodies, are needed to study protein expression patterns, sub-cellular localization, and post-translational modifications in cells and tissues. Traditionally, producing an antibody to a particular protein requires immunization of animals and recovery of antibodies from their sera. In contrast, newer methods have been developed that involve the generation of large libraries of either recombinant antibodies or other affinity scaffolds that are engineered to bind target molecules, and then screening these libraries in vitro for binding moieties having desired binding properties.
Phage display a widely used method for selecting binding molecules from recombinant antibody libraries (Lee et al., 2004. Bivalent antibody phage display mimics natural immunoglobulin. J. Immunol. Methods 284: 119-132; Marks et al., 1991. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222: 581-597; Schofield et al., 2007. Application of phage display to high throughput antibody generation and characterization. Genome Biol. 8: R254; Sheets et al., 1998. Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc. Natl. Acad. Sci. USA 95: 6157-6162). Since no immunization steps are required, comprehensive phage-antibody libraries permit in vitro targeting of antigens which are known to be toxic or possess low antigenicity in vivo. In addition, the DNA encoding the antibody is accessible in phage display, providing not only the antibody sequence, but facilitating cloning and engineering of the protein in many ways. High affinity phage antibodies have been selected against numerous cellular proteins and small molecules (Hoogenboom et al., 1998. Antibody phage display technology and its applications. Immunotechnology 4: 1-20).
Traditional methods of phage display panning involve the selection of phage display libraries against purified antigen immobilized to a solid surface. These methods may not be directly applicable to some antigens, such as membrane proteins, which may require native environments to achieve a particular structure (Deller et al., 2000. Cell surface receptors. Curr. Opin. Struct. Biol. 10, 213-219; Tate, C. G. 2001. Overexpression of mammalian integral membrane proteins for structural studies. FEBS Lett. 504, 94-98). As an alternative to selection against single protein products, several groups have panned phage libraries directly against affected tissue or whole cells (Watters et al., 1997. An optimized method for cell-based Phage display panning. Immunotechnology 3: 21-39; Nie et al., 2002. Identification of tumor-associated single-chain Fv by panning and screening antibody phage library using tumor cells. World J. Gastroenterol. 8: 619-623; Williams et al., 2002. Generation of anti-colorectal cancer fab phage display libraries with a high percentage of diverse antigen-reactive clones. Comb. Chem. High Throughput Screen 5: 489-499; Zhang et al., 2001. Neuroblastoma tumor cell-binding peptides identified through random peptide phage display. Cancer Lett. 171: 153-164; Tur et al., 2001. An anti-GD2 single chain Fv selected by phage display and fused to Pseudomonas exotoxin A develops specific cytotoxic activity against neuroblastoma derived cell lines. Int. J. Mol. Med. 8: 579-584; Hegmans et al., 2002. A model system for optimising the selection of membrane antigen-specific human antibodies on intact cells using phage antibody display technology. J. Immunol. Methods 262: 191-204; Labrijn et al., 2002. Novel strategy for the selection of human recombinant Fab fragments to membrane proteins from a phage-display library. J. Immunol. Methods 261: 37-48; Shadidi et al., 2001. An anti-leukemic single chain Fv antibody selected from a synthetic human phage antibody library. Biochem. Biophys. Res. Commun. 280: 548-552). Since cell-surface antigens may have complex conformational constraints, such as multiple membrane-spanning domains or protein interaction domains, in addition to glycosylation and other post-translational modifications, whole cell biopanning eliminates certain concerns regarding target protein structure, allowing selection against the extracellular regions of receptors in their native conformations. Such protocols can be employed, e.g., with live or fixed cells (Shukla et al., 2005. Phage display selection for cell-specific ligands: development of a screening procedure suitable for small tumor specimens. J. Drug Targeting 13: 7-18; Jakobsen et al., 2007. Phage display derived human monoclonal antibodies isolated by binding to the surface of live primary breast cancer cells recognize GRP78. Cancer Research 67: 9507; Wang et al., 2006. Selection of CC Chemokine receptor 5-binding peptide from a phage display peptide library. Biosci. Biotechnol. Biochem. 70, 2035-2041; Hoffman et al., 2004. In vivo and ex vivo selections using phage-displayed libraries. In Phage Display: A practical approach, Lowman, H. B., Clackson, T., Eds., Oxford University Press, New York, N.Y., 171-192). Since the plasma membrane is extremely complex, with diverse protein and carbohydrate structures that may complicate affinity selections using phage display libraries, subtractive panning against similar but not identical cells may be used to limit the recovery of binding moieties interacting with non-target cell surface molecules (Palmer et al., 1997. Selection of antibodies to cell surface determinants on mouse thymic epithelial cells using a phage display library. Immunology 91, 473; Popkov et al., 2004. Isolation of human prostate cancer cell reactive antibodies using phage display technology. J. Immunol. Methods 291, 137; Siva et al., 2008. Selection of anti-cancer antibodies from combinatorial libraries by whole-cell panning and stringent subtraction with human blood cells. J. Immunol. Methods 330, 109-119).
Despite the advantages of whole cell panning for the isolation of antibodies against cell-surface markers, conventional cell-based biopanning approaches can suffer from several limitations (Hoffman et al., 2004. Phage display: A practical approach. eds. T. Clackson and H. Lowman. Oxford University Press, Oxford, United Kingdom, p. 171-192). Each round of biopanning typically requires a relatively large number of cells (105-107), making the technique difficult to apply to small cell populations harvested from organs and tissues. In addition, the wash steps are often inefficient at removing nonspecifically or weakly bound phage. Repeated stringent washes, on the other hand, can lead to loss of cells and, consequently, potential antibody-displaying phage that are bound to them.